A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

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Abstract
Three methods of plasmid delivery to mesophyll protoplasts of Nicotiana tabacum cv. Xanthi have been evaluated. Specifically; a) chemically stimulated uptake of isolated plasmid, b) delivery of plasmid encapsulated in liposomes, and c) fusion of plasmid-containing spheroplasts, were combined with divalent cation (Ca2+ and Mg2+) or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol (PVA)] treatments. The quantity and quality of plasmid associated with intact protoplasts, was assessed by DNA-DNA blot hybridisation analysis, following stringent washing to separate intact protoplasts from non-viable protoplasts and debris. Treatments which increased association of plasmid with protoplasts decreased protoplast viability. Optimum association of plasmid with protoplasts, in the context of acceptable loss of viability, was achieved when protoplasts were interacted with either naked plasmid or liposomeencapsulated DNA in the presence of 15% w/v PEG 6000, or with Escherichia coli spheroplasts containing chloramphenicol-amplified plasmid in the presence of 25% w/v PEG 6000. Divalent cations did not stimulate significant plasmid delivery without unacceptable loss of protoplast viability. Strategies to further increase the efficiency of plasmid delivery are discussed.